In-hive trap and attractant composition for the control of the small hive beetle, Aethina tumida

ABSTRACT

A Small Hive Beetle trap, which replaces the bottom board of beehives, includes a frame having three walls and a top surface, a trap plate, an entry means, and a trapping means. The trapping means provides a dark environment attractive to small hive beetles and contains a small hive beetle attractant made from pollen dough and inoculated with yeast that produces small hive beetle attracting volatiles.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to a Beehive System that includes a small hivebeetle trap and to a lure for capturing small hive beetles found inhoneybee hives. It also relates to methods for using the system, thetrap, and the lure.

2. Description of the Related Art

Apiculture is a vital component of modern agriculture. Currently thereare more then 2.5 million honeybee producing colonies in the UnitedStates with an estimated value of the beekeeping and pollinationindustries of about 14.6 billion dollars per year. Honeybees,specifically Apis mellifera L., are highly efficient foragers onnumerous flowering plants, including agricultural crops. Plantpollination by the honeybee is the most commonly recognized means ofcrop pollination. Beekeepers are challenged with diseases and pestswithin hives such as the tracheal and varroa mites in the United Statesand destructive diseases such as foulbrood disease. A beehive generallyhas a fixed top and bottom and includes an exclusion device to keep thequeen bee isolated. The bottom board is generally a rectangular shapedbox or frame upon which the rest of the beehive is stacked. The bottomboard includes a gap on or in one of the four upstanding walls throughwhich bees enter and exit the beehive while keeping out other largerinsects, mice, and other rodents.

The small hive beetle is an exotic pest that originates from SouthAfrica and was found in Florida in 1998. It has now spread throughoutthe eastern and mid-western United States, causing considerable damageto honey bee colonies and negatively impacting the beekeeping industry(Morse and Calderone, Bee Culture, Volume 128, 1-15, 2000). Insub-Saharan Africa, the beetle's native home, they are not considered amajor pest of honey bees (Lundie, Sci. Bull. 220, Union of South Africa,Department of Agriculture and Forestry, 30 pages, 1940), because instrong colonies, worker honey bees encapsulate groups of small hivebeetles with propolis (Neumann et al., Naturwissenschaften, Volume 88,2140216, 200). However in the United States, where honey bee coloniesare predominantly of European origin, small hive beetles have beenreported to damage both weak and strong colonies (Sanford, Apis, Volume16, 1-5, 1998). Defense mechanisms have yet to be demonstrated inEuropean bees.

Damage to honey bee colonies in the United States is caused mainly bythe beetle larvae, which feed on honey, pollen, and brood. In addition,larval excrement tends to ferment honey, rendering it unsuitable forhuman consumption. In highly infested colonies, where larval feeding isextensive, bees generally leave. The beetle can multiply to huge numberswithin infested colonies where it eats brood, destroys combs, and ifuncontrolled ultimately destroys the hives. If the infestation issufficiently heavy, the beetles may cause bees to abandon their hive. Instored combs and honey awaiting extraction, the beetle larvae may tunnelthrough the combs of honey, feeding and defecating, causingdiscoloration and fermentation of the honey. The small hive beetle willthrive in tropical, semi-tropical and temperate climates. It is anopportunist and a scavenger, taking advantage of situations that suitits reproduction. Adult beetles lay eggs in irregular masses in crevicesor brood combs containing pollen or brood. Each female beetle is capableof laying an enormous number of eggs which can lead to a heavyinfestation in a single generation. Beetle larvae mature from egg topupae in about 10-16 days. The larvae require a diet of honey and pollento fully develop. Beetle larvae also feed on stored comb and supers ofhoney in honey houses, especially frames containing pollen. The beetleis a major problem when honey supers are allowed to remain in the beehive for long periods prior to extraction. Wax cappings, set asidefollowing honey extraction, may also become beetle infested. Bee broodrearing stops when beetle infestation reaches high levels in the colony.

Currently small hive beetles are controlled using good management and anumber of techniques that can reduce the impact of the beetle.Relocating hives may break the beetle's life cycle. Pesticide containingproducts such as CheckMite+™ strips and GardStar® (MannLake Ltd.,Hackensack, Minn.) have been recommended for hive beetle control. TheCheckMite+™ is an inside-hive treatment while the GardStar® is a soildrench.

U.S. Pat. No. 6,468,129 (Griffith, Oct. 22, 2002) discloses a bottomboard that has a screen permanently installed in the bottom board thatcovers the full expanse between the upstanding walls so that mites inthe hive have to pass through the screen and come in contact with asub-board that is removable from the bottom board through a rear access.The subboard may optionally include a sticky coating to trap the mitesthat pass through the screen.

U.S. Pat. No. 4,682,380 (Martin, Jul. 28, 1987) discloses a bottom boardwhich is flat at its base with three upstanding walls to support theremainder of the beehive with a gap provided between two of theupstanding walls to allow bees to enter and exit the hive.

Suazo et al., (Apidologie, Volume 34, 525-533, 2003) report that therehas been no attempt to identify the cues attracting small hive beetlesto honey bee hives. They state that Elzen et al. (Apidologie, Volume 31,361-366, 1999) reported that small hive beetles are attracted to acombination of honey, pollen, and adult bees. They also reported thatsmall numbers of beetles were captured in traps baited with adult beesbut not in traps baited with honey and pollen, or brood alone.

Various forms of small hive beetle control are known, however thereremains a need in the art for reliable control methods and traps forcontrolling small hive beetles. The present invention, as describedbelow, is different from related art control methods, lures, and traps.

SUMMARY OF THE INVENTION

It is therefore an object of the present invention to provide a trap,and attractant for controlling small hive beetles in beehives.

Another object of the present invention is to provide a trap for abeehive that includes a frame having three walls and a top surfacewherein an interior of two of said walls includes a means for supportinga trap plate, a trap plate, at least one entry means, and a trappingmeans.

A further object of the present invention is to provide a trap for abeehive wherein the top surface of the frame has an opening covered witha mesh screen which allows insects the size of a small hive beetle andits larvae to drop through towards the entry means and excludeshoneybees from exiting the hive through the screen.

A still further object of the present invention is to provide a trap fora beehive wherein at least one entry means includes a plurality ofgraduated tunnel-shaped entries having openings at either end wherein aproximal opening is larger than a distal opening.

Another object of the present invention is to provide a trap for abeehive wherein the proximal opening of the entry means is of a size toallow an insect the size of a small hive beetle and its larvae to crawlin and the distal opening is of a size to prevent an insect the size ofa small hive beetle and its larvae from crawling back out of thetrapping means.

A further object of the present invention is to provide a trap for abeehive wherein the trapping means has a removable top which is attachedto the trap plate using a rubber gasket.

A still further object of the present invention is to provide a trap fora beehive wherein the trapping means includes an attractant.

Another object of the present invention is to provide an attractant forsmall hive beetles that includes pollen and beetle vectored yeastwherein the attractant is fermented in the presence of moisture until itreleases small hive beetle attracting volatiles.

A still further object of the present invention is to provide anattractant wherein the pollen is natural pollen, a pollen substitute, ora mixture of the two.

A still further object of the present invention is to provide anattractant that includes pollen and yeast strain NRRL Y-30722.

Another object of the present invention is to provide yeast strainNRRL-3722.

Further objects and advantages of the present invention will becomeapparent from the following description.

Deposit of the Microorganisms

Yeast sp. NRRL Y-27634 has been deposited under the provisions of theBudapest Treaty on Mar. 15, 2004 with the U.S.D.A. Agricultural ResearchService Patent Culture Collection (National Center for AgriculturalUtilization Research, 1815 N. University Street, Peoria, Ill. 61604).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a drawing showing a top view of trap 10 showing trap top 24and trap plate 26 including frame 22, mesh screen 25 a, center opening25, trap plate opening 27 and entry means 30.

FIG. 2 is a drawing showing the top view of trap 10 showing trap top 24with frame 22, and center opening 25 with mesh screen 25 a.

FIG. 3 is a drawing showing the bottom view of frame 22 including sidewalls 21, front wall 23, top 24, center opening 25 with mesh screen 25a, and means for removably supporting trap plate 29.

FIGS. 4 a and 4 b are drawings: FIG. 4 a shows top of trap plate 26including trap plate opening 27 and entry means 30 and FIG. 4 b showsthe container portion of trapping means 28 which in this figure is thebottom portion of a Tupperware egg tray.

FIG. 5 is a drawing showing the bottom view of tray plate 26 with rubbergasket 31, entry means 30, and cover for trapping means 32.

FIGS. 6A and 6B are graphs showing wind tunnel responses of differentsexes of the small hive beetle to volatiles released from the lure fedon by (4A) 100 males, 4-8 weeks old; and (4B) 100 females, 4-8 weeks oldcompared to control (n=3 replicates, N=25 beetles/replicate). Bars withdifferent letters are statistically significant (P<0.05, LSD test).

FIG. 7 is a graph showing the number of beetles trapped between June andSeptember at a beeyard in High Springs, North Central Florida.

FIG. 8 is a graph showing the percentage of small hive beetles trappedbetween June and September at a beeyard in High Springs, North CentralFlorida.

FIG. 9 is a graph showing the effect of baited and unbaited in-hivetraps on small beetle captures at a beeyard in High Springs, NorthCentral Florida. Males-B, Females-B=males and females captures in baitedtraps. Bars with different letters are statistically significant(P<0.05, LSD test).

FIG. 10 is a graph showing effect of baited and unbaited in-hive trapson small hive beetle captures after switching traps attached to hives atthe beeyard in High Springs, North Central Florida during the summer.Males-B, Females-B=males and females captured in baited traps. Bars withdifferent letters are statistically significant (P<0.05, LSD test).

FIG. 11 is a graph showing wind tunnel responses of different sexes ofsmall hive beetles to volatiles released from the lure compared tocontrol (n=3 replicates, N=25 beetles/replicate). Bars with differentletters are statistically significant (P<0.05, LSD test).

FIG. 12 is a graph showing the effect of different lures on trap captureof the small hive beetle. Lure 1 has 50 grams of lure inoculated byfeeding males; Lure 2 has 9 grams of lure inoculated with NRRL Y-27634;control is six wet cotton balls. Bars with different letters arestatistically significant (P<0.05, LSD test).

DETAILED DESCRIPTION OF THE INVENTION

It is desirable to provide a beehive structure that eliminates or atleast reduces the incidence and effects of small hive beetleinfestation. The present invention, Trap 10, is employed as an isolationor separation device for small hive beetles infesting beehives. Trap 10is shown in FIGS. 1-5. Trap 10 includes frame 22, top 24, trap plate 26,and trapping means 28 (FIGS. 1 and 4 b). Frame 22 is constructed to fitthe external measurements of a wooden beehive. The beehive is placed ontop of trap 10. Trap 10 is the bottom board of the hive. Trap 10includes frame 22 which is made up of a front wall 23 and two side walls21; each approximately 2 inches in width (FIG. 3). Side walls 21 have ontheir inside surface, means 29 for removably supporting trap plate 26.Means 29 can be supports attached to the interior surface of side walls21. Means 29 can also be slots formed in the interior surface of walls21 and run the entire length of each of walls 21. Trap plate 26 has anopening 27 which can be of any size and is preferably rectangular inshape. Attached permanently or removably to the underside of plate 26,covering opening 27, is entry means 30 which allow beetles and larvae toenter trapping means 28, but not to exit (FIGS. 4 a and 5). Means 30 hasa plurality of graduated tunnel-shaped entries having two openings ateither end wherein the proximal opening is larger than the distalopening. The distal opening facing trapping means 28 is large enough fora small hive beetle or larvae to crawl through and drop into trappingmeans 28 but the distal opening is also small enough so that the insectscannot enter and return to the hive.

In one embodiment of the present invention (FIGS. 1-5), entry means 30is at least one PCR 96-well plate having conical-shaped wells (FIGS. 3and 5). The bottom tips of the wells are snipped off to form about a ⅛inch opening in each well for the beetles and larvae to pass through toenter trapping means 28. The bottom openings of the plate are of a sizeto allow the beetles and larvae to squeeze through to enter trappingmeans 28 but small enough that they cannot exit out of means 28.

Trapping means 28 is removably attached to the underside of trap plate26 (FIGS. 4 b and 5). Trapping means 28 can be any container with 4 sidewalls, a base, and a removable top 32 that is attached to trap plate 26so that beetles and larvae cannot escape from means 28 by crawlingbetween means 28 and trap plate 26. The top of trapping means 28 isattached to trap plate 26 using a rubber gasket 31 to seal all edges sothat beetles and/or larvae cannot escape. The removable coverfacilitates the replacement of bait and the removal of insects.Attachment of means 28 is well within the ordinary skill in the art.Minimum height of side walls of means 28 is at least about 4 inches.Trapping means 28 is opaque to provide a dark environment that isattractive to small hive beetles. In one embodiment of the presentinvention, trapping means 28 is an egg plastic storage container havinga removable cover, such as sold by Tupperware. The container is paintedblack to provide an opaque trapping means. The removable top 32 for theegg plastic storage container 28 is permanently affixed to bottom 26a oftrap plate 26 using adhesive and rubber gasket 31 allowing the containerto be removable to-replenish the bait and remove the trapped insects.The removable top 32 of the container has at least one opening 33 sothat the insects can drop into the means 28 after crawling through theentry means 30. The at least one opening 33 in the cover coincides inshape and size to that of entry means 30. Holes can be drilled into thebottom of means 28 at its shorter sides allow for drainage of rain waterwhich can flood the means 28 and destroy the attractant.

Affixed to frame 22 is a top 24 (FIGS. 1-3). Top 24 is sized to be flushwith the outer edges of frame 22 and is affixed using any means withinthe ordinary skill in the art, such as for example, nails, screws,adhesive, staples, clips, etc. Top 24 has a center opening 25 which canbe of any size or shape and equal in dimension to opening 27 in trapplate. It is preferred that the opening be rectangular in shape. Opening25 is covered with mesh screen material 25 a which has a mesh size thatallows small hive beetles to drop through but does not allow honeybeesto exit the hive through the screen. Mesh screen 25 a can be of anymaterial with ⅛ inch nylon mesh screen is preferred.

The small hive beetle lure or attractant of the present invention ismade up of a cake of moist pollen dough upon which male or female orlarvae of beetles have fed for three days. This results in naturalinoculation of the pollen dough with beetle vectored fungus.Fermentation of the pollen dough over time results in release ofvolatile chemicals that attract the small hive beetles. The dough ismade by combining either natural pollen and/or commercially availablepollen substitutes such as, for example, Bee-Pro® (Mannlake BeeSupplies, Hackensack, Minn.), with honey or corn syrup in approximatelya 1:3 ratio by weight. Moisture in the pollen dough is provided by dampcotton balls/wicks placed randomly into the container holding the dough.

In another embodiment of the invention, the pollen dough is prepared bymixing the pollen as described above with about 3 ml of novel yeastspecies strain Y-27634, NRRL Y-30722 in water, deposited under theBudapest Treaty. The ratio of pollen to yeast is about 10⁶ cells ofyeast per gram of pollen dough prior to the three day incubation periodbefore placing in traps. Mixing is accomplished by applying the aqueoussuspension of yeast to the surface of the pollen.

Strain Y27634 was isolated from small hive beetle larvae and adultinsects. The beetles were removed from an infested hive and surfacesterilized with about 70% ethanol for about 30 seconds. The insects werethen rinsed twice with sterile water. The beetles were then homogenizedin sterile water to form a suspension. The suspension was streaked ontoSabouraud dextrose agar plus 1% yeast extract (SDAY). Honey samples werealso collected from the infested hive and plated for isolation on SDAY.Inoculated plates were incubated at about 31° C. for approximately 1-3days. Individual colonies were selected and sub-cultured on SDAY.Isolates were inoculated into Durham tubes containing autoclaved beepollen broth tubes and incubated at about 31° C. for approximately 5days. Bee pollen broth is about a 1% aqueous suspension of bee pollen.The gas-producing larval isolate NRRL Y27634 produced a colonymorphology characteristic of the majority of yeast colonies observed inthe initial isolation. The yeast isolate was plated on pollen agar(about 1% pollen plus 1.5% agar), Lee's agar, Czapek-Dox broth, M40Yagar (a high sucrose, osmotic-stress medium), and moistened sterilizedpollen. Inoculated plates were incubated at about 28° C. for about 7-10days.

In operation, fermentation of either embodiment of the lure results inthe release of volatile chemicals including: acetaldehyde, ethanol,ethyl acetate, 2-methyl-1-propanol, 3-methyl-1-butanol,2-methyl-1-butanol, 3-methyl-1-butanol acetate, 2-heptanone, hexylacetate, octanal, nonanal, decanal, methyl benzoate, and relatedstraight chain and branched chain saturated alcohols and their esterswhich are highly attractive to small hive beetles. Furthermore, trappingmeans 28 is opaque and provides a dark environment that is attractive tosmall hive beetles and beetle larvae. Beetles move through screen 25 aand drop onto and into entry means 30 and traverse through theconical-shaped entries finally dropping into trapping means 28 whichcontains the pollen dough containing the attractive yeast. Once intrapping means 28, beetles remain because of the conical nature of theholes of entry means 30, and the illuminated contrast provided by theopaque trapping means 28. These conditions do not allow the beetles andlarvae to escape back into the beehive. Additionally, the pollen doughserves as food for the beetles and larvae and as an egg laying trap forfemales. Emerging larvae continue to feed on the pollen dough and thenmove out into the moist cotton balls where they attempt to pupate. Thus,all eggs, larvae, pupae, and adult beetles remain in the trap and cannot escape back into the hive. Females can lay many hundreds of eggs soeliminating a single female eliminates large numbers of beetle larvaefrom a hive. Trapping means 28 is cleaned by removing the containerportion and disposing of the insects. The insects in the trap can bekilled by placing the insects in a container such as a bucket filledwith soapy water, or the pollen dough can contain at least one insectgrowth regulator, conventional toxicant such as coumaphos for example,natural anti-insect agents applied in a carrier with or withoutadditives such as surfactants including glycerol, Tween 20®, etc.

The following examples illustrate the use of the invention. They areintended to further illustrate the invention and are not intended tolimit the scope of the invention as defined by the claims.

EXAMPLE 1

The Small Hive Beetle attractant of the present invention was preparedby mixing about 70 grams of powdered pollen made by grinding pollengrains in a household blender, about 560 grams of the commercial pollensubstitute BeePro® (MannLake, supra) in about a 1:8 ratio of powderedpollen: pollen substitute, and about 1 liter honey. To prepare the lure,the honey is warmed to about 60° C. After warming, the powdered pollenand pollen substitute were gradually added to the honey while stirringto obtain pollen dough. The dough was then allowed to cool to roomtemperature. The dough was then moistened with about 100 ml of distilledwater through a wick suspended form a vial inserted into the dough.Approximately 300 adult beetles, preferably males or virgin females,were introduced into the dough to feed and to inoculate it with theiryeast cells. The container is covered with plastic wrap or aluminum foilheld in place by a rubber band and several pin holes are punched in thetop. The container is left at room temperature for at least 3 days.Mated females can also inoculate the dough but they tend to lay eggs inthe dough. Yeast-inoculated dough from mated females has to be frozenfor a time sufficient to kill any eggs that they may lay which isusually about 3-4 hours. The frozen dough must be thawed prior to use.After about 3-7 days of feeding, the lure becomes inoculated and isready for use in a trapping system for trapping small hive beetles. Thelure can be stored at least about −20° if not used immediately.

EXAMPLE 2

Attractive activity of the lure of the present invention was tested in ahorizontal dual choice wind-tunnel (about 1.85×0.66×0.66 M) according tothe procedure described in Suazo et al (Apidologie, Volume 34, 525-533,2003; herein incorporated by reference in its entirety) and Torto et al.(Apidologie, 2004; submitted). Wind speed was set at about 0.2 m/sinside the wind tunnel. Two 34-watt fluorescent tubes about 4 feet longwere placed about 0.2 m above the wind-tunnel to provide illumination.Temperature in the bioassay room was maintained at about 27° C., with arelative humidity of about 40-60%. Tests were carried out between 6 pmand midnight, when beetle flight activity was at its peak. Odor sourceswere about 9 grams of the lure, as described above in Example 1, and asimilar weight of the pollen dough without the yeast. Each was placed ina 2 liter glass jar outside the wind-tunnel.

A stream of purified air is passed through each chamber at a flow rateof about 0.5 L/minute downwind from the odor source. Responding beetleswere captured in traps made out of plastic vials (25 dram, BioQuip,Gardena, Calif.), fitted with a screen cone with about a 5 mm opening.This opening allowed beetles to enter the vial but not exit. Tests werereplicated three times and beetles were used only once and thendiscarded. For each replicate the number of beetles responding wasrecorded for about 15 minutes. The position of odor sources was switchedbetween replicates to minimize positional bias. Beetles were deprived offood and water for one day prior to bioassays.

Results presented in FIGS. 6A and 6B clearly show that significantlymore small hive beetles were lured into traps releasing volatiles fromthe lure—71% males, 31% females—than to control traps 5% males, 6%females.

EXAMPLE 3

The lure described above in Example 1 was tested in a PVC pipe trap(about 34 cm long×9 cm OD) at a beeyard (about 30 m long×30 m wide) inHigh Springs, North Central Florida in the summer. The beeyard had 35mating nucs mounted on a wooden platform about 30 cm for the ground, and4 double storey full hives arranged in a semi-circle at the edge ofcleared field surrounded by pine and oak trees. Baited and unbaitedtraps were tested. The baited trap consisted of the PVC pipe trapcontaining about 50 grams of the moistened lure, with the unbaited traplaced with moisture only. Moisture was released into the trap through abraided cotton wick (Richmond Dental, Charlotte, N.C.) fitted throughabout a 0.5 cm hole in the cap of two plastic capped vials (BioQuip,supra) each filled with about 15 ml of distilled water. Baited andunbaited traps were hung on a T-shaped pole of about 1.5×1.5 m in thewoods, with poles distributed around the hives to fit the semi-circularpattern of the hives with six replicates. Intertrap distance on a polewas about 1 meter, while interpole and the distance of the poles fromthe hives were all about 7.5 m. Traps were set up at 4:00 pm in theafternoon and the number of beetles trapped was checked every 3-4 daysat about 11:00 am. The lure was replaced every 14 days. Results in FIG.7 clearly show that the lure is effective as a tool for monitoring smallhive beetle populations. The results also show that over 80% of thesmall hive beetles were captured between July and August (FIG. 8). Thesmall hive beetle male-female ratio in the traps was approximately1-1.5:1 during the test period, showing that the lure is effective intrapping both sexes of the beetle.

EXAMPLE 4

Lure activity was tested in combination with the In-Hive trap of thepresent invention at the same beeyard as in Example 2 in aSeptember-October period for about 27 days. Trap captures of beetlesfrom five baited and unbaited (control) traps were compared. The baitedtrap contained approximately 50 grams of moistened lure as describedabove in Example 1, while the unbaited trap was laced with moisturewhich was provided in both traps by six wet cotton balls. The number ofbeetles trapped every other day was recorded. On about day 17, thebaited and unbaited traps were switched on the hives and hive captureswere similarly recorded for a further 10 days. Trap captures for thebaited and unbaited traps were pooled separately every other day tocompute for the percentage of beetles captured (p). The data weretransformed to arcsin √{square root over (p)}, before differences amongmeans were tested for significance by ANOVA with SAS Version 8.0.Captures of the small hive beetle in the traps were significantlydifferent, and were higher by about 7-fold in the baited traps thancaptures in unbaited traps (FIGS. 9 and 10). Trap captures of males andfemales in the baited traps before and after switching traps showed nosignificant difference, confirming results obtained above in theexperiment with hanging traps (Example 2 above) that the lure iseffective in attracting both sexes of the beetle.

EXAMPLE 5

Strain Y27634 was isolated from small hive beetle larvae and adultinsects. The beetles were removed from an infested hive and surfacesterilized with about 70% ethanol for about 30 seconds. The insects werethen rinsed twice with sterile water. The beetles were then homogenizedin sterile water to form a suspension. The suspension was streaked ontoSabouraud dextrose agar plus 1% yeast extract (SDAY). Honey samples werealso collected from the infested hive and plated for isolation on SDAY.Inoculated plates were incubated at about 31° C. for approximately 1-3days. Individual colonies were selected and sub-cultured on SDAY.Isolates were inoculated into Durham tubes containing autoclaved beepollen broth tubes and incubated at about 31° C. for approximately 5days. Bee pollen broth is about a 1% aqueous suspension of bee pollen.The cultures were subcultured and volatile chemicals collected andtested for attractant qualities. The volatiles were tested with beetlesin bioassays. NRRL Y27634 was the only colony which produced attractivevolatiles. The gas-producing larval isolate NRRL Y27634 produced acolony morphology characteristic of the majority of yeast coloniesobserved in the initial isolation. The yeast isolate was plated onpollen agar (about 1% pollen plus 1.5% agar), Lee's agar, Czapek-Doxbroth, M40Y agar (a high sucrose, osmotic-stress medium), and moistenedsterilized pollen. Inoculated plates were incubated at about 28° C. forabout 7-10 days.

EXAMPLE 6

Pollen dough was prepared from pollen collected by bees. About 9 gramsof freshly collected pollen obtained from pollen traps at the entranceof a hive was autoclaved to produce sterile pollen. It was theninoculated with about 10⁶ cells of yeast strain NRRL Y-27634 per gram ofpollen. The yeast was added to the sterilized pollen as a suspension inabout 3 ml of water. This mixture is incubated at about 31° C. for about3-5 days or until there is a noticeable fruity odor. About 3 ml of honeyis added after the incubation period since the honey will prevent yeastgrowth if added prior to incubation.

Attractive activity of the lure was tested in a horizontal dual choicewind-tunnel of about 1.85×0.66×0.66 m as described above in Example 2.Wind speed was set at about 0.2 m/second inside the wind tunnel. Two34-Watt fluorescent tubes about 4 feet long were placed about 2 metersabove the wind-tunnel to provide illumination. Temperature in thebioassay room was maintained at about 27° C., with relative humidity atabout 40-60%. Tests were carried out between 19:00 and 24:00 hours whenbeetle activity was at its peak. Odor sources were about 9 grams of thelure and a similar weight without the active components, each placed ina 2 liter glass jar outside the wind-tunnel. A stream of purified airwas passed through each chamber at a flow rate of approximately 0.5liters/minute. Twenty five adult male or female small hive beetles ofabout 4-8 weeks in age were released simultaneously from a plasticholding vial (25 dram, BioQuip, Gardena, Calif.), about 1.5 metersdownwind from the odor source. Responding beetles were captured in trapsmade out of plastic vials (25 dram, BioQuip, supra) fitted with a screencone with about a 5 mm opening. This opening allowed the beetles toenter the vial but not exit. Tests were replicated three times andbeetles were used only once. For each replicate, the number of beetlesresponding was recorded for about 15 minutes. The position of odorsources was switched between replicates to minimize positional bias.Beetles were deprived of food and water for one day prior to bioassays.Results are shown in FIG. 11. FIG. 11 clearly shows that significantlymore small hive beetles were lured into traps releasing volatiles fromthe lure with about 73% males and 31% females captured compared tocontrol traps which capture about 5% males and about 6% females.

EXAMPLE 7

Attractive activity of small hive beetle lures, described above inExample 5, in combination with Trap 10 of the present invention, wastested at a beeyard in Gainesville, Fla. in July and August. Trapcaptures were compared for three different lures. Trap A contained thelure prepared as in Example 1 above. About 50 grams of the moistenedlure was placed in means for holding beetles 28 of device 10. Trap Bcontains about 9 grams of the moistened lure of Example 5 and Trap Ccontained six wet cotton balls which provided moisture to the trap. Thenumber of beetles trapped every week during the experimental period wasrecorded and the lure was replaced weekly. Trap captures of the baitedand control traps were pooled every week to compute for the percentageof beetles captured (p). The data were transformed to arcsin √{squareroot over (p)}, before differences among means were tested forsignificance by ANOVA with SAS Version 8.0. Significantly more smallhive beetles were captured from the hive baited with the lure inoculatedwith small hive beetle-yeast isolate (Example 5) than from the hivebaited with the lure prepared from feeding beetles (Example 1). Thecontrol trap was significantly less effective than either of the othertreatments in trapping small hive beetles. Results are shown in FIG. 12.

The foregoing detailed description is for the purpose of illustration.Such detail is solely for that purpose and those skilled in the art canmake variations therein without departing from the spirit and scope ofthe invention.

INDEX OF THE ELEMENTS

-   10. Trap-   21. Side Wall-   22. Frame-   23. Front Wall-   24. Top-   25. Center Opening-   25 a. Mesh Screen-   26. Trap Plate-   26 a. Trap Plate Bottom-   27. Trap Plate Opening-   28. Trapping Means-   29. Means for Removably Supporting Tray Plate-   30. Entry Means-   31. Rubber Gasket-   32. Removable Top-   33. Removable Top Opening

1. An insect trap for a beehive comprising: a frame having three wallsand a top surface wherein an interior of two of said walls includesmeans for supporting a trap plate, a trap plate operatively andremovably connected to said frame walls, at least one entry meansoperatively connected to a beehive and said trap plate, and a trappingmeans operatively connected to said entry means.
 2. The trap of claim 1wherein said top surface of said frame has an opening covered with amesh screen of a size to allow small insects to drop through towardssaid entry means and to exclude honeybees from exiting hive through saidscreen.
 3. The trap of claim 2 wherein said trap plate has an openingwhich is operatively connected to said entry means and said opening inthe top surface of said frame.
 4. The trap of claim 1 wherein said atleast one entry means includes a plurality of graduated tunnel-shapedentries having two openings at either end wherein one is a proximalopening which is larger than a distal opening.
 5. The entries of claim 4wherein said proximal opening is of a size to allow an insect the sizeof a small hive beetle or its larva to crawl in and the distal openingis of a size to prevent an insect the size of a small hive beetle or itslarva to crawl back out of said trapping means.
 6. The trap of claim 1wherein said trapping means includes a removable top which is attachedto said trap plate using a rubber gasket.
 7. The trap of claim 6 whereinsaid trapping means is opaque to provide a dark environment wherein saiddark environment is attractive to said insects.
 8. The trap of claim 6wherein said trapping means includes an attractant.
 9. The trap of claim8 wherein said attractant comprises pollen dough inoculated with yeastwhich produces small hive beetle attracting volatiles.
 10. The trap ofclaim 9 wherein said yeast is Yeast strain NRRL Y-30722.
 11. Anattractant comprising pollen, honey, and a small hive beetle vectoredfungus wherein said attractant is fermented in the presence of moistureuntil the release of volatiles attractive to small hive beetles.
 12. Theattractant of claim 11 wherein said pollen is selected from the groupconsisting of natural pollen, pollen substitute, and mixtures thereof.13. An attractant comprising pollen and a yeast producing small hivebeetle attracting volatiles.
 14. The attractant of claim 13 wherein saidyeast is yeast species NRRL Y-30722.
 15. A method for trapping smallhive beetles comprising: a. replacing the bottom board of a beehive witha trap wherein said trap includes, a frame having three walls and a topsurface wherein an interior of two of said walls includes means forsupporting a trap plate, a trap plate operatively and removablyconnected to said frame walls, at least one entry means operativelyconnected to a beehive and said trap plate, and a trapping meansoperatively connected to said entry means, and b. placing an attractantin said trapping means.
 16. The method of claim 15 wherein said topsurface of said trap has an opening covered with a mesh screen of a sizeto allow small insects to drop through towards said entry means and toexclude honeybees from exiting the hive through said screen.
 17. Themethod of claim 16 wherein said trap plate of said trap has an openingoperatively connected to said entry means and said opening on the topsurface of said frame.
 18. The method of claim 15 wherein said at leastone entry means includes a plurality of graduated tunnel-shaped entrieshaving two openings at either end wherein one is a proximal opening of alarger size than a distal opening.
 19. The method of claim 18 whereinsaid proximal openings are of a size to allow an insect the size of asmall hive beetle or its larva to crawl in and the distal opening is ofa size to prevent an insect the size of a small hive beetle or its larvafrom exiting said trapping means.
 20. The method of claim 15 whereinsaid trapping means includes a removable top which is attached to saidtrap plate using a rubber gasket.
 21. The method of claim 20 whereinsaid trapping means is opaque to provide a dark environment wherein saiddark environment is attractive to said insects.
 22. The method of claim20 wherein said trapping means includes an attractant.
 23. The method ofclaim 23 wherein said attractant comprises pollen dough inoculated withyeast which produces small hive beetle attracting volatiles.
 24. Themethod of claim 23 wherein said yeast is yeast strain NRRL Y-30722. 25.A strain of yeast isolated from small hive beetles wherein said yeaststrain produces small hive beetle attracting volatiles.
 26. The strainof claim 25 having the characteristics of deposited strain NRRL Y-30722.27. An isolated strain of yeast producing small hive beetle attractingvolatiles designated NRRL Y-30722.